This invention relates to gene therapy whereby modified steroid receptors regulate the expression of genes within tissue.
Intracellular receptors are a superfamily of related proteins that mediate the nuclear effects of steroid hormones, thyroid hormone and vitamins A and D (Evans, Science 240:889-895 (1988)). The cellular presence of a specific intracellular receptor defines that cell as a target for the cognate hormone. The mechanisms of action of the intracellular receptors are related in that they remain latent in the cytoplasm or nuclei of target cells until exposed to a specific ligand (Beato, Cell 56:335-344 (1989); O""Malley, et al., Biol. Reprod. 46:163-167 (1992)). Interaction with hormone then induces a cascade of molecular events that ultimately lead to the specific association of the activated receptor with other proteins or regulatory elements of target genes. The resulting positive or negative effects on regulation of gene transcription are determined by the cell-type and promoter-context of the target gene.
In the case of steroid hormones and steroid receptors, such complexes are responsible for the regulation of complex cellular events, including activation or repression of gene transcription. For example, the ovarian hormones, estrogen and progesterone, are responsible, in part, for the regulation of the complex cellular events associated with differentiation, growth and functioning of female reproductive tissues. Likewise, testosterone is responsible for the regulation of complex cellular events associated with differentiation growth and function of male reproductive tissues.
In addition, these hormones play important roles in development and progression of malignancies of the reproductive endocrine system. The reproductive steroids estrogen, testosterone, and progesterone are implicated in a variety of hormone-dependent cancers of the breast (Sunderland, et al., J. Clin. Oncol. 9:1283-1297 (1991)), ovary (Rao, et al., Endocr. Rev. 12:14-26 (1991)), endometrium (Dreicer, et al., Cancer Investigation 10:27-41, (1992)), and possibly prostate (Daneshgari, et al., Cancer 71:1089-1097 (1993)). In addition, the onset of post-menopausal osteoporosis is related to a decrease in production of estrogen (Barzel, Am. J. Med. 85:847-850 (1988)).
In addition, corticosteroids are potent and well-documented mediators of inflammation and immunity. They exert profound effects on the production and release of numerous humoral factors and the distribution and proliferation of various cellular components associated with the immune and inflammatory responses. For example, steroids are able to inhibit the production and release of cytokines (IL-1, IL-2, IL-3, IL-6, IL-8, TNF-xcex1, IFN-xcex3), chemical mediators (eicosinoids, histamine), and enzymes (MMPs) into tissues, and directly prohibit the activation of macrophages and endothelial cells. Due to the global down-regulation of these physiological events, corticosteroids have a net effect of suppressing the inflammatory response and have therefore been used extensively to treat a variety of immunological and inflammatory disorders (rheumatoid arthritis, psoriasis, asthma, allergic rhinitis, etc.).
Besides the therapeutic benefits, however, there are some severe toxic side effects associated with steroid therapy. These include peptic ulcers, muscle atrophy, hypertension, osteoporosis, headaches, etc. Such side effects have hindered the utilization of steroids as therapeutic agents.
In general, the biological activity of steroid hormones is mediated directly by a hormone and tissue-specific intracellular receptor. Ligands are distributed through the body by the hemo-lymphatic system. The hormone freely diffuses across all membranes but manifests its biological activity only in those cells containing the tissue-specific intracellular receptor.
In the absence of ligand, the inactive steroid hormone receptors such as the glucocorticoid (xe2x80x9cGRxe2x80x9d), mineral corticoid (xe2x80x9cMRxe2x80x9d), androgen (xe2x80x9cARxe2x80x9d) progesterone (xe2x80x9cPRxe2x80x9d) and estrogen (xe2x80x9cERxe2x80x9d) receptors are sequestered in a large complex consisting of the receptor, heat-shock proteins (xe2x80x9chspxe2x80x9d) 90, hsp70 and hsp56 and other proteins as well. Smith, et al., Mol. Endo. 7:4-11 (1993). The cellular localization of the physiologically inactive form of the oligomeric complex has been shown to be either cytoplasmic or nuclear. Picard, et al., Cell Regul. 1:291-299 (1992); Simmons, et al., J. Biol. Chem. 265:20123-20130 (1990).
Upon binding its agonist or antagonist ligand, the receptor changes conformation and dissociates from the inhibitory heteroligomeric complex. Allan, et al., J. Biol. Chem. 267:19513-19520 (1992); Allan, et al., P.N.A.S. 89:11750-11754 (1992). In the case of GR and other related systems such as AR, MR, and PR, hormone binding elicits a dissociation of heat shock and other proteins and the release of a monomeric receptor from the complex. O""Malley, et al., Biol. Reprod. 46:163-167 (1992). Studies from genetic analysis and in vitro protease digestion experiments show that conformational changes in receptor structure induced by agonists are similar but distinct from those induced by antagonists. Allan, et al., J. Biol. Chem. 267:19513-19520 (1992); Allan, et al., P.N.A.S. 89:11750-11754 (1992); Vegeto, et al., Cell 69:703-713 (1992). However, both conformations are incompatible with hsp-binding.
Following the conformation changes in receptor structure, the receptors are capable of interacting with DNA. Studies suggest that the DNA binding form of the receptor is a dimer. In the case of GR homodimers, Tsai, et al., Cell 55:361-369 (1988), this allows the receptor to bind to specific DNA sites in the regulatory region of target gene promoters. Beato, Cell 56:335-344 (1989). These short nucleotide sketches are arranged as palindromic, inverted or repeated repeats. Id. Specificity is determined by the sequence and the spacing of the repeated sequences. Umesono, et al., Cell 57:1139-1146. Following binding of the receptor to DNA, the hormone is responsible for mediating a second function that allows the receptor to interact specifically with the transcription apparatus. Such interaction could either provide positive or negative regulation of gene expression, i.e., steroid receptors are ligand-binding transcription factors, capable of not only activating but also repressing the expression of specific genes. Studies have shown, however, that repression does not require DNA binding.
For instance, when bound to their intracellular receptors, corticosteroids can affect the transcription of a variety of genes whose products play key roles in the establishment and progression of an inflamed situation. Such genes include those encoding for cytokines, chemical mediators and enzymes. Transcription of these genes can be repressed or activated depending on the transcription factors and/or regulatory sequences controlling the expression of the gene. Presently there are numerous reports documenting the effect of glucocorticoid on the expression of various genes at the transcriptional level.
In particular, the glucocorticoid receptor is a member of a family of ligand-dependent transcription factors capable of both positive and negative regulation of gene expression (Beato, FASEB J. 5:2044-2051 (1991); Pfahl, Endocr. Rev. 14:651-658, (1993); Schule, et al., Trends Genet. 7:377-381 (1991)). In its inactivated form, the GR is part of a large heteromeric complex which includes hsp90 as well as other proteins (Denis, et al., J. Biol. Chem. 262:11803-11806 (1987); Howard, et al., J. Biol. Chem. 263:3474-3481 (1988); Mendel, et al., J. Biol. Chem. 261:3758-3763 (1986); Rexin, et al., J. Biol. Chem. 267:9619-9621 (1992); Sanchez, et al., J. Biol. Chem. 260:12398-12401 (1985)), and hsp56 (Lebea, et al., J. Biol. Chem. 267:4281-4284 (1992); Pratt, J. Steroid Biochem. Mol. Biol. 46:269-279 (1993); Rexin, J. Biol. Chem. 267:9619-9621 (1992); Sanchez, J. Biol. Chem. 265:22067-22070 (1990); Yem, J. Biol. Chem. 267:2868-2871, (1992)). Binding of agonist stimulates receptor activation, dissociation from hsp90 and the other proteins (Denis, et al., Nature 333:686-688 (1988); Sanchez, et al., J. Biol. Chem. 262:6986-6991 (1987)), and nuclear translocation, prerequisites for both transactivation and transrepression.
Cloning of several members of the steroid receptor superfamily has facilitated the reconstitution of hormone-dependent transcription in heterologous cell systems and facilitated delineation of the GR activation and repression mechanisms. Subsequently, in vivo and in vitro studies with mutant and chimeric receptors have demonstrated that steroid hormone receptors are modular proteins organized into structurally and functionally defined domains. Deletion mutants of the GR have determined that the transactivation domain is located at the N-terminal amino acid sequence positioned between amino acids 272 and 400. Jonat, et al., Cell 62:1189-1204 (1990). A well defined 66 amino acid DNA binding domain (xe2x80x9cDBDxe2x80x9d) has been identified and studied in detail, using both genetic and biochemical approaches. Lucibello, et al., EMBO J. 9:2827-2834 (1990). The ligand or hormone binding domain (xe2x80x9cLBDxe2x80x9d), located in the carboxyl-terminal portion of the receptor, consists of about 300 amino acids. Kerppola, et al., Mol. Cell. Biol. 13:3782-3791 (1993). The LBD has not been amenable to detailed site-directed mutagenesis, since this domain appears to fold into a complex tertiary structure, creating a specific hydrophobic pocket which surrounds the effector ligand when bound. This feature creates difficulty in distinguishing among amino acid residues that affect the overall structure of the LBD domain from those involved in a direct contact with the ligand. The LBD also contains sequences responsible for receptor dimerization, nuclear localization, hsp interactions and transactivation sequences of the receptor. Fuller, et al, FASEB J. 5:3092-3099 (1991).
The mechanism of gene activation is far better understood than that of repression. For transactivation, a ligand-induced conformational change, comparable to that inferred to be necessary for activation of the progesterone (Allan, et al., Proc. Natl. Acad. Sci. USA 35 89:11750-11754 (1992)) and estrogen (Beekman, et al., Mol. Endocrinol. 7:1266-1274 (1993)) receptors, is required for efficient activation of the transcription activating function of the receptor (Hollenberg and Evans, Cell 55:899-906 (1988); Webster, et al., Cell 54:199-207, (1988)). Furthermore, the conformational change is required for interaction of the receptor with other components of the transcription apparatus. Transactivation is mediated by a receptor dimer bound to a glucocorticoid response element (xe2x80x9cGRExe2x80x9d). Such transactivation occurs exclusively by homodimerization. This is mainly achieved by a region in the second zinc finger of the receptor known as the D-loop. Umesono, et al., Cell 57:1139-1146 (1989); Dahlman-Wright, et al., J. Biol. Chem. 266:3107-3112 (1991). The resulting homodimers then bind to the palindromic GRE to initiate the transcriptional activation process. Evans, Science 240:889-895 (1988); Cato, et al.; J. Steroid Biochem. Mol. Biol. 43:63-68 (1992).
Transrepression, on the other hand, appears to be mediated by the monomeric form of the receptor through interactions with other transcriptional factors, including AP-1 and NFK-B, preventing them from carrying out their function as transcriptional activators. Hoeck, et al., EMBO J. 13:4087-4095 (1994). Studies also show transrepression by the dimeric form of the receptor. In the case of the monomeric pathway, studies suggest that AP-1 prevents hormone-dependent activation of GR-regulated promoters through a mutually inactive complex formed either by a direct protein-protein interaction of the receptor and AP-1 or through a third partner. Miner, et al., Cell Growth Differ. 2:525-530 (1991); Pfahl, Endocrine Rev. 14:651-658 (1993). Such transrepression of AP-1 and NFK-B mediated by the monomeric form of the receptor depends on the presence of the DNA binding domain. It does not depend on the ability of the receptor to bind DNA. In the case of the dimeric form of the receptor, several studies suggest mechanisms for such GR-mediated transrepression include GR binding to a sequence overlapping a cis-acting element for another trans-acting factor, thereby displacing it from, or preventing its binding to, its cognate element (Akerblom, et al., Science 241:350-353 (1988); Drouin, et al., Mol. Cell. Biol. 9:5305-5314 (1989); Oro, et al., Cell 55:1109-1114, (1988); Stromstedt, et al., Mol. Cell. Biol. 11:3379-3383, (1991)).
As noted above, GR-mediated transrepression attributed to direct or indirect interaction of the GR with other trans-acting factors, results in inhibition of their activity and/or ability to bind to DNA (Celada, et al., J. Exp. Med. 177:691-698 (1993); Diamond, et al., Science 249:1266-1272 (1990); Gauthier, et al., Embo J. 12:5089-5096 (1993); Jonat, et al., Cell 62:1189-1204 (1990); Kutoh, et al., Mol. Cell Biol. 12:4955-4969 (1992); Lucibello, et al., Embo J. 9:2827-2834 (1990); Ray, et al., Proc. Natl. Acad. Sci. USA 91:752-756 (1994); Schule, et al., Cell. 62:1217-1226 (1990); Tverberg, et al., J. Biol. Chem. 267:17567-17573 (1992); Yang-Yen, et al., Cell 62:1205-1215 (1990); Lucibello, et al., EMBO J. 9:2827-2834 (1990)). These models require ligand binding to stimulate receptor activation, dissociation from hsp90, and nuclear translocation. It is not clear whether these mechanisms are dependent on the same ligand-induced conformational change needed for transactivation. However, a transactivation-defective mutant represses the AP-1 dependent promoter suggesting that the transactivation function of the receptor is not required for the repression of AP-1 activity. Yang-Yen, et al., Cell 62:1205-1215 (1990). Furthermore, similar studies also suggest that the transactivation function of the receptor is not required for the repression of NFK-B activity.
In attempts to decipher the transrepression mechanism, studies have reviewed the role of the bound ligand in GR-mediated repression of AP-1-responsive genes containing a tetradecanoyl phorbol acetate (xe2x80x9cTPAxe2x80x9d) response element. Repression of these genes has been proposed to be the result of the direct interaction of the GR with c-Jun (Diamond, et al., Science 249:1266-1272 (1990); Lucibello, et al., EMBO J. 9:2827-2834 (1990); Schule, et al., Cell 62:1217-1226 (1990); Touray, et al., Oncogene 6:1227-1234 (1991); Yang-Yen, et al., Cell 62:1205-1215 (1990) ) or c-Fos (Kerppola, et al., Mol. Cell. Biol. 13:3782-3791 (1992)) which are components of the AP-1 transcription complex. The GR DNA-binding domain is necessary for this interaction, since most mutations in this domain result in the loss of repressor activity in vivo (Diamond, et al., Science 249:1266-1272 (1990); Jonat, et al., Cell 62:1189-1204 (1990); Lucibello, et al., EMBO J. 9:2827-2834 (1990); Schule, et al., Cell 62:1217-1226 (1990); Yang-Yen, et al., Cell 62:1205-1215 (1990)).
The DNA-binding domain is also necessary for inhibition of in vitro transcription from the collagenase promoter and inhibition of Jun-Fos heterodimer binding to the collagenase TPA response element (Mordacq, et al., Genes Dev. 3:760-769 (1989)). However, deletion or truncation of the ligand-binding domain also results in a significant loss of repressor activity (Jonat, et al., Cell 62:1189-1204 (1990); Schule, et al., Cell 62:1217-1226 (1990); Yang-Yen, et al., Cell 62:1205-1215 (1990)), suggesting that the ligand-binding domain may contribute to, or modulate, the inhibition of AP-1 activity.
Further studies examining the role of the ligand in GR-mediated transrepression of the collagenase promoter found efficient receptor-mediated transrepression with ligand-free mutant GR in which the first cysteine residue of the proximal zinc finger was replaced with tyrosine. Liu, et al., Mol. Cell. Bio. 15:1005-1013 (1995). Such studies suggest that neither retention of the ligand nor direct binding of the receptor to DNA is required, i.e., that transrepression of AP-1 activity by GR is ligand independent.
Applicants have determined that it is useful to construct modified steroid hormone receptors which regulate the expression of nucleic acid sequences. Specifically, these modifications allow control of the transactivation and transrepressing functions of the modified steroid hormone receptor. Such modifications allow the receptors to bind various ligands whose structures differ dramatically from the naturally-occurring ligands. This includes the binding of non-natural ligands, anti-hormones and non-native ligands.
These modifications are generated in the ligand binding domain of the GR and eliminate the ability of the GR to bind its natural ligand. These modified steroid receptors exhibit normal transactivation and transrepression activity; however, stimulation of such activity occurs via activation by a non-natural and exogenously or endogenously applied ligand. Modifications are also generated in the ligand binding domain of the PR and eliminate the ability of PR to bind its natural ligand. Replacement of the GR binding domain with the modified PR binding domain allows the stimulation of GR responsive gene expression via non-natural ligands.
Other modifications to the GR ligand binding domain in conjunction with modifications to the DNA binding domain of GR eliminate the ability of steroid hormones to initiate transactivation by its natural ligand. Instead, such modifications allow the modified receptor to bind non-natural ligands and stimulate the transrepression regulation of gene expression but not transactivation. Likewise, using the same ligand binding domain modification in conjunction with modifications to the transregulatory domain allows the modified receptor to bind non-natural ligands and stimulate transactivation but not transrepression of gene expression.
Other modifications remove the ligand binding domain completely to create a constitutively active steroid receptor. Such modifications cause continual transactivation and transrepression effects on the regulation of gene transcription. In addition, modifications that selectively eliminate either transactivation or transrepression functions are incorporated into the constitutively active steroid receptor thereby constitutively transrepressing or transactivating gene expression. Furthermore, other modifications use a ligand binding domain which recognizes its natural ligand or if modified recognizes a non-natural ligand, but is fused with a DNA binding domain and transregulatory domains not associated normally with the ligand binding domain. Such a construct is capable of regulating the expression of a gene not normally associated with the ligand binding domain in a wild type receptor protein.
These modified receptors can be expressed by specially designing DNA expression vectors to control the level of expression of recombinant gene products. The steroid receptor family of gene regulatory proteins is an ideal set of such molecules. These proteins are ligand activated transcription factors whose ligands can range from steroids to retinoids, fatty acids, vitamins, thyroid hormones and other presently unidentified small molecules. These compounds bind to receptors and either activate or repress transcription.
These receptors are modified to allow them to bind various ligands whose structure is either naturally occurring or differs from naturally occurring ligands. By screening receptor mutants, receptors can be selected that respond to ligands which do not activate the host cell endogenous receptor. Thus, regulation of a desired transgene can be achieved using a ligand which binds to and regulates a customized receptor. This occurs only with cells that have incorporated and express the modified receptor.
Taking advantage of the abilities of the modified steroid hormone receptor to effect regulation of gene expression, these gene constructs can be used as therapeutic gene medicines. These modified receptors are useful in gene therapy where the level of expression of a gene, whether transactivation or repression, is required to be controlled. The number of diseases associated with inappropriate production or responses to hormonal stimuli highlights the medical and biological importance of these constructs.
The properties of the modified steroid hormone receptors allow the deleterious effects of steroids to be avoided while maintaining their therapeutic benefits. In particular, administration of steroids causes toxicity problems. The deleterious effects of steroids can be attributed to the in vivo transactivation or transrepression of certain genes. These toxic effects may well be the result of both transactivation and transrepression, or be primarily attributable to one of them. The present invention features the use of modified GR molecules as gene medicines for the replacement of steroid therapy. These synthetic receptors retain functions similar to those of the endogenous receptors, but by responding to alternative ligands, eliminate some of the toxic side effects attributable to currently used steroid therapy.
This ability of the GR constructs to avoid steroid toxicity but still exhibit therapeutic effects allows the constructs to be used for treating numerous diseases, including arthritis, asthma, senile dementia or Parkinson""s disease. Furthermore, the constructs can be used for preventing or treating diseases in which inappropriate production or responses to hormonal stimuli exists, e.g., hormone-dependent cancers of the breast, ovary, endometrium, prostate, and post-menopausal osteoporosis. The constructs also can be used in conjunction with co-transfected expression vectors so as to operate as a gene switch. For detailed description of gene switch, see, U.S. application Ser. No. 07/939,246, Vegeto et al., and U.S. Pat. No. 5,364,791, Vegeto et al., the whole of which (including drawings) are both hereby incorporated by reference.
In addition, the constructs above can be used for gene replacement therapy in humans and for creating transgenic animal models used for studying human diseases. The transgenic models can be used as well for assessing and exploring novel therapeutic avenues to treat effects of chemical and physical carcinogens and tumor promoters. The above constructs can also be used for distinguishing steroid hormone receptor antagonists and steroid hormone receptor agonists. Such recognition of antagonist or agonist activity can be performed using cells transformed with the above constructs.
In a first aspect, the present invention features a modified glucocorticoid receptor fusion protein. The fusion protein receptor is GR with its ligand-binding domain replaced with a mutated PR ligand-binding domain. This fusion protein is capable of being activated by the binding of a non-natural ligand but not by natural or synthetic glucocorticoid or other natural or synthetic steroids. The fusion protein includes a glucocorticoid receptor region which comprises a DNA binding domain and transregulatory domains. The transregulatory domains are capable of transactivating or transrepressing glucocorticoid responsive gene expression. Such mutations and fusion proteins can be created from different receptors and from different species, and still accomplish the same physiological effect. Thus, the present invention is not limited to glucocorticoid receptors nor to the species herein.
In addition to the glucocorticoid receptor region, the fusion protein also includes a mutated progesterone ligand binding region which is capable of binding a non-natural ligand. The mutated ligand binding region is mutated by deletion of about 42 to 54 carboxyl terminal amino acids of a progesterone receptor ligand binding domain. The mutated progesterone receptor ligand binding region comprises about amino acids 640 through 891 of a progesterone receptor. Other embodiments comprise amino acids 640-917 while other embodiments comprise amino acids 640-920. One skilled in the art will recognize that various mutations can be created to achieve the desired function.
The term xe2x80x9cfusion proteinxe2x80x9d as used herein refers to a protein which is composed of two or more proteins, or fragments thereof, occurring separately in nature. The combination can be between complete amino acid sequences of the protein as found in nature, or fragments thereof. In the case of the glucocorticoid-progesterone fusion protein receptor, the fusion protein is composed of portions of the glucocorticoid receptor and the progesterone receptor. This combination can include the complete amino acid sequence of each protein or fragments thereof. For example, the glucocorticoid-progesterone fusion protein may include the ligand binding domain of progesterone and the DNA binding domain and transregulatory domains of the glucocorticoid receptor. This is only an example and not meant to be limiting.
In addition to the above, other fusion proteins can be constructed. A useful construct includes a fusion protein comprising: (1) a ligand binding domain which binds endogenous ligand, and (2) a DNA binding domain and/or transregulatory domains not naturally associated with the ligand binding domain. Such a construct allows the regulation of expression of other genes, whether activation or repression, which are not normally regulated by the ligand binding domain. A person skilled in the art will recognize that there are other possible variations of the above fusion protein that are within the scope of the present invention.
The term xe2x80x9cnon-natural ligandxe2x80x9d as used herein refers to compounds which can normally bind to the ligand binding domain of a receptor but are not the endogenous ligand. The receptor is not exposed to the ligand unless it is exogenously supplied. This also includes ligands or compounds which are not normally found in animals or humans. Non-natural also includes ligands which are not naturally found in the specific organism (man or animal) in which gene therapy is contemplated. These ligands activate receptors by binding to the modified ligand binding domain. Activation can occur through a specific ligand-receptor interaction whether it is through direct binding or through association in some form with the receptor.
xe2x80x9cNatural ligandxe2x80x9d as used here refers to compounds which normally bind to the ligand binding domain of a receptor and are endogenous. The receptor in this case is exposed to the ligand endogenously. Natural ligands include steroids, retinoids, fatty acids, vitamins, thyroid hormones, as well as synthetic variations of the above. This is meant to be only an example and non-limiting.
The term xe2x80x9cligandxe2x80x9d as referred to herein means any compound which activates the receptor, usually by interaction with the ligand binding domain of the receptor. Ligand includes a molecule or an assemblage of molecules capable of specifically binding to a modified receptor. The term xe2x80x9cspecifically bindingxe2x80x9d means that a labelled ligand bound to the receptor can be completely displaced from the receptor by the addition of unlabelled ligand, as is known in the art.
Examples of non-natural ligands and non-native ligands include the following: 11xcex2-(4-dimethylaminophenyl)-17xcex2-hydroxy-17xcex1-propinyl-4,9-estradiene-3-one (RU486 or Mifepripeestone); 11xcex2-(4-dimethylaminophenyl)-17xcex1-hydroxy-17xcex2-(3-hydroxypropyl)-13xcex1-methyl-4,9-gonadiene-3-one (ZK98299 or Onapristone); 11xcex2-(4-acetylphenyl)-17xcex1-hydroxy-17xcex1-(1-propinyl)-4,9-estradiene-3-one (ZK112993); 11xcex1-(4-dimethylaminophenyl)-17xcex2-hydroxy-17xcex1-(3-hydroxy-1(Z)-propenyl-estra-4,9-diene-3-one (ZK-98734); (7xcex2,11xcex2,17xcex2)-11-(4-dimethylaminophenyl)-7-methyl-4xe2x80x2,5xe2x80x2-dihydrospiro[ester-4,9-diene-17,2xe2x80x2(3xe2x80x2H)-furan]-3-one (Org31806); (11xcex2,14xcex2,17xcex1)-4xe2x80x2,5xe2x80x2-dihydro-11-(4-dimethylaminophenyl)-[spiroestra-4,9-diene-17,2xe2x80x2(3xe2x80x2H)-furan]-3-one (Org31376); 5xcex1-pregnane-3,2-dione.
The term xe2x80x9cbindingxe2x80x9d or xe2x80x9cboundxe2x80x9d as used herein refers to the association, attaching, connecting, or linking through covalent or non-covalent means, of a ligand, whether non-natural or natural, with a corresponding receptor. The ligand and receptor interact at complementary and specific within sites on a given structure. Binding includes, but is not limited to, components which associate by electrostatic binding, hydrophobic binding, hydrogen binding, intercalation or forming helical structures with specific sites on nucleic acid molecules.
The term xe2x80x9cglucocorticoid receptorxe2x80x9d refers to a steroid hormone receptor which responds to a glucocorticoid ligand. The glucocorticoid receptor is part of the steroid hormone receptor superfamily which are known steroid receptors whose primary sequence suggests that they are related to each other. Representative examples of such receptors include the estrogen, progesterone, Vitamin D, chicken ovalbumin upstream promoter transfactor, ecdysone, Nurr-1 and orphan receptors, glucocorticoid-xcex1, glucocorticoid-xcex2, mineralocorticoid, androgen, thyroid hormone, retinoic acid, and retinoid X. These receptors are composed of DNA binding domains, ligand binding domains, as well as transregulatory domains.
The glucocorticoid receptor is a ligand-dependent transcription factor capable of both positive and negative regulation of gene expression. Interaction of the receptor with a ligand induces a cascade of molecular events that ultimately lead to the specific association of the activated receptor with regulatory elements of target genes. In an inactive form such receptors form a large complex comprising the receptor, heat shock proteins and other proteins.
The term xe2x80x9cglucocorticoid receptor regionxe2x80x9d refers to a fragment or part of the complete glucocorticoid receptor as defined above. A glucocorticoid receptor region may retain complete or partial activity of the natural receptor protein. For example, a glucocorticoid receptor region might contain only the DNA binding domain and the transregulatory domains and not the ligand binding domain, or vice versa. This is only an example and not meant to be limiting.
The term xe2x80x9cligand binding domainsxe2x80x9d or xe2x80x9cligand binding regionxe2x80x9d as used herein refers to that portion of a steroid hormone receptor protein which binds the appropriate hormone or ligand and induces a cascade of molecular events that ultimately leads to the specific association of the activated receptor with regulatory elements of target genes. This includes, but is not limited to, the positive or negative effects on regulation of gene transcription. Binding of ligand to the ligand binding domain induces a conformation change in the receptor structure. The conformational change includes the dissociation of heat shock proteins and the release of a monomeric receptor from the receptor complex, as well as a different tertiary or 3-dimensional structure. The conformational change that occurs is specific for the steroid receptor and ligand that binds to the ligand binding domain.
For example, for glucocorticoid receptors, the conformation change that occurs when glucocorticoid hormone binds allows homodimerization, i.e., dimerization between two identical GR molecules. However, heterodimerization can occur with other steroid receptors, i.e., dimerization with two molecules such as GR and ER. Such dimerization allows the receptor to bind with DNA or induce the regulatory effect by binding other transcription factors.
The term xe2x80x9cDNA binding domainxe2x80x9d as used herein refers to that part of the steroid hormone receptor protein which binds specific DNA sequence in the regulatory regions of target genes. This domain is capable of binding short nucleotide stretches arranged as palindromic, inverted or repeated repeats. Such binding, will activate gene expression depending on the specific ligand and the conformational changes due to such ligand binding. For repression, DNA binding is not needed.
The term xe2x80x9ctransregulatory domainxe2x80x9d as used herein refers to those portions of the steroid hormone receptor protein which are capable of transactivating or transrepressing gene expression. This would include different regions of the receptor responsible for either repression or activation, or the regions of the receptor responsible for both repression and activation. Such regions are spacially distinct. The above is only an example and meant to be non-limiting. For transrepression, this domain under one mechanism is involved with dimerization which in turn causes a protein/protein interaction to prevent or repress gene expression. Such regulation occurs when the receptor is activated by the ligand binding to the ligand binding domain. The conformational change of the receptor is capable of forming a dimer with a discrete portion of the transregulatory domain to repress gene expression. In addition, repression can occur through a monomeric form of the receptor, however, DNA binding is not necessary (see below).
The terms xe2x80x9ctransactivation,xe2x80x9d xe2x80x9ctransactivate,xe2x80x9d or xe2x80x9ctransactivatingxe2x80x9d refer to a positive effect on the regulation of gene transcription due to the interaction of a hormone or ligand with a receptor causing the cascade of molecular events that ultimately lead to the specific association of the activated receptor with the regulatory elements of the target genes. Transactivation can occur from the interaction of non-natural as well as natural ligands. Agonist compounds which interact with steroid hormone receptors to promote transcriptional response can cause transactivation. Such positive effects on transcription include the binding of an activated receptor to specific recognition sequences in the promoter of target genes to activate transcription. The activated receptors are capable of interacting specifically with DNA. The hormone- or ligand-activated receptors associate with specific DNA sequences, or hormone response elements, in the regulatory regions of target genes. Transactivation alters the rate of transcription or induces the transcription of a particular gene(s). It refers to an increase in the rate and/or amount of transcription taking place.
The terms xe2x80x9ctransrepress,xe2x80x9d xe2x80x9ctransrepressionxe2x80x9d or xe2x80x9ctransrepressingxe2x80x9d as used herein refer to the negative effects on regulation of gene transcription due to the interaction of a hormone or ligand with a receptor inducing a cascade of molecular events that ultimately lead to the specific association of the activated receptor with other transcription factors such as NFK-B or AP-1. Transrepression can occur from the interaction of non-natural as well as natural ligands. Antagonist and agonist compounds which interact with steroid hormone receptor can cause transrepression. Once the ligand binds to the receptor, a conformational change occurs. Transrepression can occur via two different mechanisms, i.e., through the dimeric and monomeric form of the receptor. Use of the monomeric form of the receptor for transrepression depends on the presence of the DNA binding domain but not on the ability of the receptor to bind DNA. Use of the dimeric form of the receptor for transrepression depends on the receptor binding response elements overlapping cis-element(s). Transrepression alters the rate of transcription or inhibits the transcription of a particular gene. Transrepression decreases the rate and/or the amount of transcription taking place.
The term xe2x80x9cprogesterone receptorxe2x80x9d as used herein also refers to a steroid hormone receptor which responds to or is activated by the hormone progesterone. Progesterone is part of the steroid hormone receptor superfamily as described above. The progesterone receptor can exist as two distinct but related forms that are derived from the same gene. The process for generation of the products may be alternate initiation of transcription, splicing differences, or transcription termination. These receptors are composed of DNA binding, ligand binding, as well as transregulatory domains. The progesterone receptor is also a ligand-dependent transcription factor capable of regulating gene expression. Interaction of the progesterone receptor with a ligand induces a cascade of molecular events that ultimately lead to the specific association of the activated receptor with regulatory elements of target genes.
The term xe2x80x9cmodified,xe2x80x9d modification,xe2x80x9d xe2x80x9cmutantxe2x80x9d or xe2x80x9cmutatedxe2x80x9d refers to an alteration of the receptor from its naturally occurring wild-type form. This includes alteration of the primary sequence of a receptor such that it differs from the wild-type or naturally-occurring sequence. The mutant steroid hormone receptor protein as used in the present invention can be a mutant of any member of the steroid hormone receptor superfamily. For example, a steroid receptor can be mutated by deletion of amino acids on the carboxyl terminal end of the protein. Generally, a deletion of from about 1 to about 120 amino acids from the carboxyl terminal end of the protein provides a mutant steroid hormone receptor useful in the present invention. A person having ordinary skill in this art will recognize, however, that a shorter deletion of carboxyl terminal amino acids will be necessary to create useful mutants of certain steroid hormone receptor proteins. Other mutations or deletions can be made in other domains of the steroid receptor of interest, such as the DNA binding domain or the transregulatory domain.
For example, a mutant of the progesterone receptor protein will contain a carboxyl terminal amino acid deletion of approximately 1 to 60 amino acids. In a preferred embodiment of the present invention, 42 carboxyl terminal amino acids are deleted from the progesterone receptor protein. Likewise, a mutation of one or more amino acids in the DNA binding domain or the transregulatory domains can change the regulation of gene expression.
One skilled in the art will recognize that a combination of mutations and/or deletions are possible to gain the desired response. This would include double point mutations to the same or different domains. In addition, mutation also includes xe2x80x9cnull mutationsxe2x80x9d which are genetic lesions to a gene locus that totally inactivate the gene product.
One example is the generation of GR constructs by incorporating mutations in the GR to produce the desired effect. This would include, but is not limited to, mutations to amino acids 421 to 481 of the rat GR to eliminate the ability of the GR to transrepress promoter constructs dependent on AP-1 and NFK-B while still retaining the ability to transactivate the expression of GRE-dependent promoter constructs. Such mutations which generate transactivation but not transrepressing activity include 1) the serine at position 425 changed to a glycine, the leucine at position 436 changed to a valine and the tyrosine and asparagine at positions 478 and 479 would be changed to leucine and glycine respectively. This is only an example and not a limitation. One skilled in the art will be well aware that other mutations can be created to provide the desired effect. Such mutations can be used in human GR constructs.
Mutations can also be generated in the D-loop of the DNA binding domain of GR that interfere with dimerization of GR. These mutations eliminate the ability of GR to transactivate but still promote transrepressing efficiently. In the case of rat GR such mutations transrepress even better than the wild type rat GR. Such mutations eliciting transrepression but not transactivation activity include the alanine at position 458 changed to a threonine, the asparagine and alanine at positions 454 and 458 changed to aspartic acid and threonine, respectively, and arginine and aspartic acid at positions 460 and 462 changed to aspartic acid and cysteine, respectively. The above mutated regions can be further and more precisely defined in humans by routine methodology, e.g., deletion or mutation analysis or their equivalent to obtain a ligand binding domain without natural ligand activity but with non-natural ligand activity. The above is only an example and meant to be non-limiting.
The term mutation also includes any other derivatives. The term xe2x80x9cderivativexe2x80x9d as used herein refers to a peptide or compound produced or modified from another peptide or compound of a similar structure. This could be produced in one or more steps. The term xe2x80x9cmodifiedxe2x80x9d or xe2x80x9cmodificationxe2x80x9d as used herein refers to a change in the composition or structure of the compound or molecule. However, the activity of the derivative, modified compound, or molecule is retained, enhanced, or increased relative to the activity of the parent compound or molecule. This would include the change of one amino acid in the sequence of the peptide or the introduction of one or more non-naturally occurring amino acids or other compounds. This includes a change in a chemical body, a change in a hydrogen placement, or any type of chemical variation. In addition, xe2x80x9canalogxe2x80x9d as used herein refers to a compound that resembles another structure. Analog is not necessarily an isomer. The above are only examples and are not limiting.
The term xe2x80x9cnucleic acid sequence,xe2x80x9d xe2x80x9cgene,xe2x80x9d xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9cnucleic acid cassettexe2x80x9d as used herein refers to the genetic material of interest which can express a protein, or a peptide, or RNA after it is incorporated transiently, permanently, or episomally into a cell. The nucleic acid can be positionally and sequentially oriented in a vector with other necessary elements such that the nucleic acid can be transcribed and, when necessary, translated into protein in the cells.
The term xe2x80x9cgenetic materialxe2x80x9d as used herein refers to contiguous fragments of DNA or RNA. The genetic material which is introduced into targeted cells can be any DNA or RNA. For example, the nucleic acid can be: (1) normally found in the targeted cells, (2) normally found in targeted cells but not expressed at physiologically appropriate levels in targeted cells, (3) normally found in targeted cells but not expressed at optimal levels in certain pathological conditions, (4) not normally found in the targeted cells, (5) novel fragments of genes normally expressed or not expressed in targeted cells, (6) synthetic modifications of genes expressed or not expressed within targeted cells, (7) any other DNA which may be modified for expression in targeted cells and (8) any combination of the above.
The term xe2x80x9cgene expressionxe2x80x9d or xe2x80x9cnucleic acid expressionxe2x80x9d as used herein refers to the gene product of the genetic material from the transcription and translation process. Expression includes the polypeptide chain translated from an mRNA molecule which is transcribed from a gene. If the RNA transcript is not translated, e.g., rRNA, tRNA, the RNA molecule represents the gene product.
The expression of the glucocorticoid-progesterone fusion protein receptor can be expressed as a cell surface, cytoplasmic or nuclear protein. By xe2x80x9ccell surface proteinxe2x80x9d it is meant that a protein is wholly or partially spanning the cell membrane when expressed and which also is exposed on the surface of the cell. By cytoplasmic protein it is meant that a protein is contained completely within the cytoplasm, and does not span the nucleus or cell surfaces. As for xe2x80x9cnuclear proteinxe2x80x9d it is meant that the protein is wholly or partially spanning the nuclear membrane when expressed and is exposed to the cell cytoplasm, or may be contained completely within the cell nucleus, not attached to the nuclear membrane and not exposed to cell cytoplasm.
A second aspect of the present invention features a modified glucocorticoid receptor protein. The glucocorticoid receptor protein contains a DNA binding domain, transregulatory domains and a mutated ligand binding domain. The modified protein is capable of binding a non-natural ligand by the mutated ligand binding domain. The mutated ligand domain is created by deleting about 2-5 carboxyl terminal amino acids from the ligand binding domain. In a preferred embodiment, the modified glucocorticoid receptor protein can be mutated by deleting amino acids 762 and 763, and substituting or altering amino acids 752 and 753, of the ligand binding domain. Substituted amino acids 752 and 753 can be changed to be both alanines.
A third aspect of the present invention features a modified glucocorticoid receptor protein. This protein contains a DNA binding domain and transregulatory domains. The transregulatory domains are capable of constitutively transactivating or transrepressing gene expression. The receptor protein is mutated by removing the ligand binding domain. As used herein the term xe2x80x9cconstitutivelyxe2x80x9d refers to the ability to continually activate or repress gene expression without the need for a ligand.
A fourth aspect of the present invention features a modified glucocorticoid receptor protein. This protein is capable of binding a non-natural ligand. The modified receptor contains a glucocorticoid receptor region which comprises a DNA binding domain, a mutated transregulatory domain and a mutated ligand binding domain. The mutated transregulatory domains are capable of transactivating gene expression but not transrepressing gene expression.
For example, the mutated ligand binding domain is mutated as described above. The rat GR mutated transregulatory domain is mutated by changing the serine at position 425 to glycine, the leucine at position 436 to valine, and the tyrosine and asparagine at positions 478 and 479 to leucine and glycine. Such mutations can be used in human GR.
A fifth aspect of the present invention features a modified glucocorticoid receptor protein which is capable of binding a non-natural ligand. The modified receptor contain a glucocorticoid receptor region which comprises a mutated DNA binding domain, transregulatory domains and a mutated ligand binding domain. The mutated DNA binding domain prevents transactivation since DNA binding is necessary for such activation. The transregulatory domains are capable of transrepressing gene expression but not transactivating gene repression. Such activity occurs upon binding of the mutated binding ligand with the non-natural ligand.
For example, the mutated ligand binding domain is mutated as described above. The rat GR mutated DNA binding domain is mutated by changing the alanine at position 458 to threonine, the asparagine and alanine at positions 454 and 458 changed to aspartic acid and threonine respectively, and the arginine and aspartic acid at positions 460 and 562 changed to aspartic acid and cysteine, respectively. Such mutations can be used in human GR.
A sixth related aspect of the invention features a nucleic acid sequence encoding one of the modified glucocorticoid receptors as discussed above, including the fusion protein receptor. The nucleic acid is the genetic material which can express a protein, or a peptide, or RNA after it is incorporated transiently, permanently or episomally into a cell.
A seventh related aspect of the present invention features a vector containing a nucleic acid sequence for the modified glucocorticoid receptors discussed above. The vectors are capable of expressing the nucleic acid transiently, permanently or episomally into a cell or tissue. In one example, the vector is a plasmid designated as pGR0403R for the constitutively active GR and pGR0385 for mutated rat GR.
The term xe2x80x9cvectorxe2x80x9d as used herein refers to a construction comprised of genetic material designed to direct transformation of a targeted cell. A vector contains multiple genetic elements positionally and sequentially oriented with other necessary elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary translated in the transfected cells. The term vector as used herein can refer to nucleic acid, e.g., DNA derived from a plasmid, cosmid, phagemid or bacteriophage, into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular proteins. The term xe2x80x9cplasmidxe2x80x9d as used herein refers to a construction comprised of extrachromosomal genetic material, usually of a circular duplex of DNA which can replicate independently of chromosomal DNA. The plasmid does not necessarily replicate.
The vector can contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector molecule can confer some well-defined phenotype on the host organism which is either selectable or readily detected. The vector may have a linear or circular configuration. The components of a vector can contain but is not limited to a DNA molecule incorporating: (1) DNA; (2) a sequence encoding a therapeutic or desired product; and (3) regulatory elements for transcription, translation, RNA processing, RNA stability, and replication.
The purpose of the vector is to provide expression of a nucleic acid sequence in cells or tissue. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence. Expression products may be proteins, polypeptides, or RNA. The nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous, constitutive, or regulated. The vector can also be used as a prokaryotic element for replication of plasmid in bacteria and selection for maintenance of plasmid in bacteria.
In the present invention the preferred vector comprises the following elements linked sequentially at an appropriate distance to allow functional expression: a promoter, a 5xe2x80x2 mRNA leader sequence, a translation initiation site, a nucleic acid cassette containing the sequence to be expressed, a 3xe2x80x2 mRNA untranslated region, and a polyadenylation signal sequence. As used herein the term xe2x80x9cexpression vectorxe2x80x9d refers to a DNA vector that contains all of the information necessary to produce a recombinant protein in a heterologous cell.
In addition, the term xe2x80x9cvectorxe2x80x9d as used herein can also include viral vectors. A xe2x80x9cviral vectorxe2x80x9d in this sense is one that is physically incorporated in a viral particle by the inclusion of a portion of a viral genome within the vector, e.g., a packaging signal, and is not merely DNA or a located gene taken from a portion of a viral nucleic acid. Thus, while a portion of a viral genome can be present in a vector of the present invention, that portion does not cause incorporation of the vector into a viral particle and thus is unable to produce an infective viral particle.
A vector as used herein can also include DNA sequence elements which enable extra-chromosomal (episomal) replication of the DNA. Vectors capable of episomal replication are maintained as extra-chromosomal molecules and can replicate. These vectors are not eliminated by simple degradation but continue to be copied. These elements may be derived from a viral or mammalian genome. These provide prolonged or xe2x80x9cpersistentxe2x80x9d expression as described below.
The term xe2x80x9cpersistent expressionxe2x80x9d as used herein refers to introduction of genes into the cell together with genetic elements which enable episomal (i.e., extrachromosomal) replication. This can lead to apparently stable transformation of the cell without the integration of the novel genetic material into the chromosome of the host cell.
xe2x80x9cStable expressionxe2x80x9d as used herein relates to the integration of genetic material into chromosomes of the targeted cell where it becomes a permanent component of the genetic material in that cell. Gene expression after stable integration can permanently alter the characteristics of the cell and its progeny arising by replication leading to stable transformation.
An eighth related aspect of the present invention features a transfected cell containing a vector which contains nucleic acid sequence for a modified glucocorticoid receptor as discussed above. As used herein the term xe2x80x9ctransfectedxe2x80x9d or xe2x80x9ctransfectionxe2x80x9d refers to the incorporation of foreign DNA into any cells by exposing them to such DNA. This would include the introduction of DNA by various delivery methods, e.g., via vectors or plasmids.
Methods of transfection may include microinjection, CaPO4 precipitation, liposome fusion (e.g., lipofection), electroporation or use of a gene gun. Those are only examples and are meant not to be limiting. The term xe2x80x9ctransfectionxe2x80x9d as used herein refers to the process of introducing DNA (e.g., DNA expression vector) into a cell. Following entry into the cell, the transfected DNA may: (1) recombine with the genome of the host; (2) replicate independently as an episome; or (3) be maintained as an episome without replication prior to elimination. Cells may be naturally able to uptake DNA. Particular cells which are not naturally able to take up DNA require various treatments, as described above, in order to induce the transfer of DNA across the cell membrane.
A ninth related aspect of the present invention features a transformed cell with a vector containing a nucleic acid sequence for a modified glucocorticoid receptor as discussed above. As used here in the term xe2x80x9ctransformedxe2x80x9d or xe2x80x9ctransformationxe2x80x9d refers to transient, stable or permanent changes in the characteristics (expressed phenotype) of a cell by the mechanism of gene transfer. Genetic material is introduced into a cell in a form where it expresses a specific gene product or alters the expression or effects of endogenous gene products.
The term xe2x80x9cstablexe2x80x9d as used herein refers to the introduction of gene(s) into the chromosome of the targeted cell where it integrates and becomes a permanent component of the genetic material in that cell. Gene expression after stable transformation can permanently alter the characteristics of the cell leading to stable transformation. An episomal transformation is a variant of stable transformation in which the introduced gene is not incorporated in the host cell chromosomes but rather is replicated as an extrachromosomal element. This can lead to apparently stable transformation of the characteristics of a cell. xe2x80x9cTransientlyxe2x80x9d as used herein refers to the introduction of a gene into a cell to express the nucleic acid, e.g., the cell express specific proteins, peptides or RNA, etc. The introduced gene is not integrated into the host cell genome and is accordingly eliminated from the cell over a period of time. Transient expression relates to the expression of a gene product during a period of transient transfection. Transient expression also refers to transfected cells with a limited life span.
Transformation can be performed by in vivo techniques as described below or ex vivo techniques in which cells are co-transfected with a vector containing a selectable marker. This selectable marker is used to select those cells which have become transformed. It is well known to those skilled in the art the type of selectable markers to be used with transformation studies. Transformation can be tissue specific to regulate expression of the nucleic acid predominantly in the tissue or cell of choice.
Transformation of the cell may be associated with production of a variety of gene products including protein and RNA. These products may function as intracellular or extracellular structural elements, ligands, hormones, neurotransmitters, growth regulating factors, enzymes, serum proteins, receptors, carriers for small molecular weight compounds, drugs, immunomodulators, oncogenes, tumor suppressors, toxins, tumor antigens, antigens, antisense inhibitors, triple strand forming inhibitors, ribozymes, or as a ligand recognizing specific structural determinants on cellular structures for the purpose of modifying their activity. Other examples can be found above in the discussion of nucleic acid cassette. The product expressed by the transformed cell depends on the nucleic acid of the nucleic acid cassette. This list is only an example and is not meant to be limiting. In the present invention the nucleic acid to be expressed is a fusion protein as referenced above, or variations thereof or any of the other receptor proteins disclosed herein.
In one embodiment the transformed cell is a muscle cell. The term xe2x80x9cmusclexe2x80x9d refers to myogenic cells including myoblasts, skeletal, heart and smooth muscle cells. The muscle cells or tissue can be in vivo, in vitro or tissue culture and capable of differentiating into muscle tissue. In another embodiment, the transformed cell is a lung cell. The term xe2x80x9clung cellxe2x80x9d as used herein refers to cells associated with the pulmonary system. The lung cell can also be in vivo, in vitro or tissue culture.
In still another embodiment, the transformed cell is a cell associated with the joints. The term xe2x80x9ccells associated with the jointsxe2x80x9d refers to all of the cellular and non-cellular materials which comprise the joint (e.g., knee or elbow) and are involved in the normal function of the joint or are present within the joint due to pathological conditions. These include material associated with: the joint capsule such as synovial membranes, synovial fluid, synovial cells (including type A cells and type B synovial cells); the cartilaginous components of the joint such as chondrocyte, extracellular matrix of cartilage; the bony structures such as bone, periosteum of bone, periosteal cells, osteoblast, osteoclast; the immunological components such as inflammatory cells, lymphocytes, mast cells, monocytes, eosinophil; other cells like fibroblasts; and combinations of the above. Once transformed these cells express the fusion protein. One skilled in the art will quickly realize that any cell is capable of undergoing transformation and within the scope of this invention.
A tenth aspect of the present invention features methods for transforming a cell with a vector containing nucleic acid encoding for a modified glucocorticoid receptor. This method includes the steps of transforming a cell in situ by contacting the cell with the vector for a sufficient amount of time to transform the cell. As discussed above, transformation can be in vivo or ex vivo. Once transformed the cell expresses the mutated glucocorticoid receptor. This method includes methods of introducing and methods of incorporating the vector. xe2x80x9cIncorporatingxe2x80x9d and xe2x80x9cintroducingxe2x80x9d as used herein refer to uptake or transfer of the vector into a cell such that the vector can express the therapeutic gene product within a cell as discussed with transformation above.
An eleventh aspect of the present invention features a method of using the modified glucocorticoid receptors discussed above. This method comprises the steps of transforming a cell with a vector containing a nucleic acid encoding for the modified glucocorticoid receptor of interest. The transformed cells are able to express the mutated glucocorticoid receptor. The receptor is capable of regulating by a non-natural ligand the expression of glucocorticoid responsive genes, whether such regulation is transactivation or transrepression. The term xe2x80x9cglucocorticoid responsive genesxe2x80x9d as used herein refers to genes whose expression is regulated by the activation of the glucocorticoid receptor. Such regulation includes both positive and negative regulation of gene expression. This also includes GRE (glucocorticoid response element) controlled genes.
This method of use includes methods of gene replacement using the fusion protein, methods of gene therapy using the fusion protein and methods of administering the fusion protein in which the same steps are used. xe2x80x9cGene replacementxe2x80x9d as used herein means supplying a nucleic acid sequence which is capable of being expressive in viva in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
The methods of use also include methods for using the modified glucocorticoid receptor to activate GRE controlled genes. Such genes can be co-transfected with the modified glucocorticoid receptors. Such co-transfection allows activated expression of the GRE controlled genes. Furthermore, the methods of use include the use of tissue specific delivery systems, and use of mRNA stability constructs.
The present invention features methods for administration as discussed above. Such methods include methods for administering a supply of polypeptide, protein or RNA to a human, animal or to tissue culture or cells. These methods of use of the above-referenced vectors comprises the steps of administering an effective amount of the vectors to a human, animal or tissue culture. The term xe2x80x9cadministeringxe2x80x9d or xe2x80x9cadministrationxe2x80x9d as used herein refers to the route of introduction of a vector or carrier of DNA into the body. The vectors of the above methods and the methods discussed below may be administered by various routes. Administration may be intravenous, intratissue injection, topical, oral, or by gene gun or hypospray instrumentation. Administration can be directly to a target tissue, e.g. direct injection into synovial cavity or cells, or through systemic delivery. These are only examples and are nonlimiting.
Administration will include a variety of methods, such as direct gene transfer into muscle tissue by liposomes, proteoliposomes, calcium phosphate-co-precipitated DNA, DNA coupled to macro-molecular complexes, DNA transporters, DNA coated to micro-projectiles, coated plasmids, direct micro-injection, as well as tissue grafting. Direct gene transfer of vectors can be administered by microinjection, electroporation, liposomes, proteoliposomes, calcium-phosphate-co-precipitation, tissue grafting, retroviral vectors, DNA coupled to macromolecular complexes, DNA transporters, gene gun and micro-projectiles. See, e.g., WO 93/18759, hereby incorporated by reference herein. The preferred embodiment is by direct injection. Routes of administration include intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal, intrathecal and/or fluid spaces.
The term xe2x80x9ceffective amountxe2x80x9d as used herein refers to sufficient vector administered to humans, animals or into tissue culture cells to produce the adequate levels of polypeptide, protein, or RNA. One skilled in the art recognizes that the adequate level of protein polypeptide or RNA will depend on the intended use of the particular vector. These levels will be different depending on the type of administration, treatment or vaccination as well as intended use.
In one embodiment of the present invention, the method of using the mutated glucocorticoid receptors discussed above uses RU486 as the non-natural ligand to regulate gene expression. This ligand is capable of binding the mutated progesterone or glucocorticoid ligand binding domain and activating the transregulatory domains of the receptor. RU486 is capable of activating or repressing the appropriate glucocorticoid responsive genes. This is only and example and not meant to be limiting. Those skilled in the art will recognize that other non-natural ligands can be used.
The method of use can regulate transactivation of glucocorticoid responsive genes or GRE controlled genes or gene constructs. In addition, the method of use can regulate transrepression of glucocorticoid responsive genes such as metalloproteinases, interleukins, cyclooxygenases, and cytokines. Although such genes respond to other stimuli, these genes are repressed by steroids. Typically, without the primary stimulant, steroids have little effect on the basal transcription of such genes. Genes such as IL-2, IL-6, IL-8, ICAM-1, VCAM-1 have been repressed by steroids. Any gene transcription depending on AP-1 or NFK-B will be repressed in the present invention.
A twelfth aspect of the present invention features a method for treating arthritis. This method includes the transformation of cells associated with the joints with the above referenced vectors. The vectors contain nucleic acid which encode for the modified glucocorticoid receptor protein. Once expressed in the cells associated with the joints, the mutated protein is capable of transactivating or transrepressing by a non-natural ligand the expression of glucocorticoid responsive genes or GRE controlled genes, including transfected GRE controlled gene constructs.
With respect to the joints, diseases which can be treated by the methods of the present invention include those diseases known to one in the art as arthritis. This includes pathophysiological conditions resulting from inflammatory processes; hypertrophy or inappropriate proliferation of cellular elements of the joint; damage to the joint; enhancement of repair, regeneration, and recovery of essential structures comprising the joint after surgery or injury; and other acquired diseases of the joints. For example, in the treatment of a pathological condition the vector with or without a formulation will be introduced into cells comprising structures of the joint by injecting a pharmacological dose of the vector with or without a formulation into a joint. The nucleic acid cassette in the vector encodes a protein, polypeptide or RNA. The vector is taken up by appropriate cells within the joint and expresses the protein, polypeptide or RNA. The preferred embodiment of this invention involves transient or persistent expression within the joint. This is preferable to stable expression since it enables adjustment of the level of expression in response to the evolution of the disease process.
Specific diseases which can be treated by administration of vectors to cells within the joint include various arthritises, avascular necrosis, or injuries requiring repair and regeneration of structures comprising the joint. The various types of arthritis which can be treated, include but are not limited to: tendinitis; bursitis; fibrositis; bone lesions; soft tissue inflammation; degenerative joint disease; traumatic disorders; neuropathic arthropathy; metabolic disorders; synovial tumors; pigmented villonodular synovitis; hemorrhagic disorders; septic disorders; crystal-induced disorders (gout); immune complex disease and vasculitis; systemic lupus erythematosus; rheumatoid arthritis; Reiter""s syndrome; psoriasis; ankylosing spondylitis; scleroderma; and arthritis of intestinal disease. In a specific embodiment of the present invention, an anti-inflammatory cytokine may be expressed including IL-4, IL-10, or TGF-xcex2.
Cells associated with fluid spaces incorporate the formulated DNA expression vector into the cell. xe2x80x9cIncorporatexe2x80x9d refers to uptake or transfer of the formulated DNA expression vector into a cell such that the formulated DNA expression vector can express the therapeutic gene product within the cell, i.e., the mutated receptor. Significantly, incorporation may involve, but does not require, integration of the DNA expression vector or episomal replication of the DNA expression vector. Incorporation in this sense includes the short term persistence of the DNA expression vector in the cell before it is eliminated by degradation or translocation to other compartments.
Incorporation includes expression of the nucleic acid cassette by cells, whether it is transient expression, persistent expression, or stable expression. xe2x80x9cTransient expressionxe2x80x9d as used herein relates to the introduction of genetic material into a cell to express specific proteins, peptides or RNA, etc. The introduced genetic material is not integrated into or replicated by the host cell genome, but is accordingly eliminated from the cell over a period of time by degradation or translocation to other compartments. These terms are defined in more detail above.
A thirteenth aspect of the present invention features a method for treating asthma. This method includes the transformation of cells associated with the lungs or pulmonary system with the above referenced vectors. The vectors contain nucleic acid which encodes the fusion protein. Once expressed in the lung cells the mutated receptor is capable of transactivating or transrepressing the expression by a non-natural ligand of the appropriate glucocorticoid responsive genes and/or GRE controlled transgenes.
In one embodiment, the above methods of treatment invoke use of RU486 as the non-natural ligand. The transactivation and transrepression can occur when the mutated glucocorticoid receptor is activated by RU486. The genes that are transrepressed or transactivated in response to ligand binding to the fusion protein are described above.
A fourteenth aspect of the present invention features a transgenic animal whose cells contain the vectors of the present invention. These cells include germ or somatic cells. Transgenic animal models can be used for understanding of molecular carcinogenesis and disease, assessing and exploring novel therapeutic avenues for effects by potential chemical and physical carcinogens and tumor promoters.
An additional preferred embodiment provides for a transgenic animal containing a modified glucocorticoid receptor vector. By xe2x80x9ctransgenic animalxe2x80x9d is meant an animal whose genome contains an additional copy or copies of the gene from the same species or it contains the gene or genes of another species, such as a gene encoding for a mutated glucocorticoid receptor introduced by genetic manipulation or cloning techniques, as described herein and as known in the art. The transgenic animal can include the resulting animal in which the vector has been inserted into the embryo from which the animal developed or any progeny of that animal. The term xe2x80x9cprogenyxe2x80x9d as used herein includes direct progeny of the transgenic animal as well as any progeny of succeeding progeny. Thus, one skilled in the art will readily recognize that if two different transgenic animals have been made each utilizing a different gene or genes and they are mated, the possibility exists that some of the resulting progeny will contain two or more introduced genes. One skilled in the art will readily recognize that by controlling the matings, transgenic animals containing multiple introduced genes can be made.